Project Summary An essential decision that germline stem cells must make is to proliferate mitotically or initiate meiotic development/gametogenesis. Disruption of this developmental switch can result in infertility and in some cases germline tumors. The C. elegans adult hermaphrodite is an important model for understanding control of the switch from germline stem cell fate to meiotic development/gametogenesis, where an outline of the process is emerging. Niche dependent GLP-1 Notch signaling promotes the stem cell fate and represses three redundant pathways that promote meiotic development/gametogenesis: the GLD-1 pathway (which represses expression of proliferation genes), the GLD-2 pathway (which promotes expression of meiotic genes), and a 3rd pathway whose existence has been revealed through genetic analysis but no gene products have been identified to date. At a cellular level, we have recently shown that the stem cell population is large and germ cells enter meiosis directly, without intervening transit-amplifying divisions. The absence of transit-amplifying divisions simplifies the analysis allowing straightforward assays to identify genes involved in repressing meiosis in stem cells and repressing proliferation at meiotic entry and is the primary reason why C. elegans is the major animal model for studying this important developmental switch. This proposal addresses three major areas in the stem cell - meiotic development/gametogenesis switch where there are large gaps in our knowledge. First, the transcriptional targets of GLP-1 signaling that promote the stem cell fate and/or repress the meiotic entry pathways are incompletely known. We propose to identify GLP-1 transcriptional targets and determine which meiotic entry pathway(s) they inhibit. Second, genes that constitute the 3rd meiotic entry pathway are unknown. Our preliminary results indicate that the SCFprom-1 ubiquitin mediated degradation complex is the 3rd pathway and we propose experiments to further test this hypothesis. Third, gld-1 mRNA translation/stability regulation is a central part of germline stem cell differentiation as expression must be repressed in stem cells and activated for meiosis; however control of GLD-1 accumulation is incompletely understood. We propose to (i) determine the mechanism of how two known GLP-1 transcriptional targets repress GLD-1 accumulation, (ii) determine the spatial and quantitative contribution of newly identified regulators of the germline stem cell differentiation in control of GLD-1 accumulation, and (iii) identify new regulators, since known regulators explain only part of the GLD-1 accumulation pattern, as a route to identify additional genes that control the developmental switch.